Synthetic receptors induce anti angiogenic and stress signaling on human first trimester cytotrophoblast cells

Article abstract of DOI:10.3390/ijerph14050517

The cytotrophoblast (CTB) cells of the human placenta have membrane receptors that bind certain cardiotonic steroids (CTS) found in blood plasma. One of these, marinobufagenin, is a key factor in the etiology of preeclampsia. Herein, we used synthetic receptors (SR) to study their effectiveness on the angiogenic profile of human first trimester CTB cells. The humanextravillous CTB cells (Sw.71) use in this study were derived from first trimester chorionic villus tissue. Culture media of CTB cells treated with ≥1 nM SR level revealed sFlt-1 (Soluble fms-like tyrosine kinase-1) was significantly increased while VEGF (vascular endothelial growth factor) was significantly decreased in the culture media (*p < 0.05 for each) The AT2 receptor (Angiotensin II receptor type 2) expression was significantly upregulated in ≥1 nM SR-treated CTB cells as compared to basal; however, the AT₁ (Angiotensin II receptor, type 1) and VEGFR-1 (vascular endothelial growth factor receptor 1) receptor expression was significantly downregulated (* p < 0.05 for each). Our results show that the anti-proliferative and anti-angiogenic effects of SR on CTB cells are similar to the effects of CTS. The observed anti angiogenic activity of SR on CTB cells demonstrates that the functionalized-urea/thiourea molecules may be useful as potent inhibitors to prevent CTS-induced impairment of CTB cells.

Full text of DOI:10.3390/ijerph14050517

International Journal of
Environmental Research
and Public Health
Article
Synthetic Receptors Induce Anti Angiogenic and
Stress Signaling on Human First Trimester
Cytotrophoblast Cells
Ahmed F. Pantho 1, Mason Price 2, AHM Zuberi Ashraf 3, Umaima Wajid 3,
Maryam Emami Khansari 4, Afsana Jahan 4, Syeda H. Afroze 5, Md Mhahabubur Rhaman 4,
Corey R. Johnson 4, Thomas J. Kuehl 3,6, Md. Alamgir Hossain 4,* and
Mohammad Nasir Uddin 3,6,7,
*
1
Department of Biochemistry, University of Texas at Austin, Austin, TX 78712, USA;
ahmedfaiaz1995@utexas.edu
2
3
Department of Biology, Angelo State University, San Angelo, TX 76904, USA; mprice15@angelo.edu
Department of Obstetrics & Gynecology, Baylor Scott & White Health, Temple/Texas A&M Health Science
Center College of Medicine, Temple, TX 76508, USA; xeonflow16@gmail.com (A.Z.A.);
Umaima.Wajid@BSWHealth.org (U.W.); Thomas.Kuehl@BSWHealth.org (T.J.K.)
Department of Chemistry & Biochemistry, Jackson State University, Jackson, MS 39217, USA;
mary.emami@yahoo.com (M.E.K.); afsanajahan01@gmail.com (A.J.);
4
md_mhahabubur.rhaman@jsums.edu (M.M.R.); corey19144@gmail.com (C.R.J.)
Medical Physiology, Texas A&M Health Science Center College of Medicine, Temple, TX 76504, USA;
Afroze@medicine.tamhsc.edu
Department of Pediatrics, Baylor Scott & White Health, Temple/Texas A&M Health Science Center College
of Medicine, Temple, TX 76508, USA; TKUEHL@sw.org
5
6
7
Internal Medicine, Baylor Scott & White Health, Temple/Texas A&M Health Science Center College of
Medicine, Temple, TX 76508, USA
*
Correspondence: alamgir.hossain@jsums.edu (M.A.H.); mohammad.uddin1@bswhealth.org (M.N.U.)
Academic Editor: William Chi-shing Cho
Received: 7 April 2017; Accepted: 28 April 2017; Published: 11 May 2017
Abstract: The cytotrophoblast (CTB) cells of the human placenta have membrane receptors that
bind certain cardiotonic steroids (CTS) found in blood plasma. One of these, marinobufagenin, is a
key factor in the etiology of preeclampsia. Herein, we used synthetic receptors (SR) to study their
effectiveness on the angiogenic profile of human first trimester CTB cells. The humanextravillous
CTB cells (Sw.71) used in this study were derived from first trimester chorionic villus tissue. Culture
media of CTB cells treated with
was significantly increased while VEGF (vascular endothelial growth factor) was significantly
≥
1 nM SR level revealed sFlt-1 (Soluble fms-like tyrosine kinase-1)
decreased in the culture media (* p < 0.05 for each) The AT2 receptor (Angiotensin II receptor
type 2) expression was significantly upregulated in
≥
1 nM SR-treated CTB cells as compared to
basal; however, the AT1 (Angiotensin II receptor, type 1) and VEGFR-1 (vascular endothelial growth
factor receptor 1) receptor expression was significantly downregulated (* p < 0.05 for each). Our
results show that the anti-proliferative and anti-angiogenic effects of SR on CTB cells are similar to
the effects of CTS. The observed anti angiogenic activity of SR on CTB cells demonstrates that the
functionalized-urea/thiourea molecules may be useful as potent inhibitors to prevent CTS-induced
impairment of CTB cells.
Keywords: angiogenic; cardiotonic steroids; cell signaling; cytotrophoblast; preeclampsia;
synthetic receptors
Int. J. Environ. Res. Public Health 2017, 14, 517; doi:10.3390/ijerph14050517
www.mdpi.com/journal/ijerph

Int. J. Environ. Res. Public Health 2017, 14, 517
2 of 15
1. Introduction
Preeclampsia (preE) [
(diastolic 90 mmHg) and proteinuria (
5–10% of pregnancies [ ]. PreE is the second leading cause of maternal and fetal morbidity and mortality
in the world [ ]. The incidence of preE has risen in the U.S. [ ]. PreE is often accompanied by
intrauterine growth restriction (IUGR) and is associated with preterm birth. Both of the latter conditions
prejudice the survival and well being of the fetus [ ]. There is no definitive therapy other than delivery.
Cardiotonic steroids (CTS) [10
11] are endogenous steroidal inhibitors of the Na+/K+ ATPase
1] is a hypertensive disorder of pregnancy characterized by hypertension
≥
≥
300 mg in 24 h) after 20 weeks of gestation. PreE occurs in
2
3–6
7,8
9
,
(NKA). Vasoconstrictor digitalis-like CTS are a second natriuretic system. In addition to
vaso-relaxant atrial natriuretic peptides [12], CTS are key factors in the regulation of blood pressure.
A number of CTS have been identified in human plasma and urine, including cardenolides (OUB)
(e.g., endogenous ouabain (EO) and digoxin) and bufadienolides (e.g., marinobufagenin (MBG),
bufalin, and resibufogenin). Binding to the CTS receptor site on the α-subunit of the NKA induces
natriuresis [3,13,14]. In addition to transport of Na+ and K+, the NKA functions as a receptor, which is
capable of transducing CTS binding into activation of intracellular protein kinases and alterations in
Ca2+ levels, ultimately altering the cell-surface expression of the NKA and Na+/H+ exchanger [15].
In this light, CTS can be viewed as a new class of hormones, rather than simply as endogenous
inhibitors of Na+ transport. Recent data support a role for CTS, specifically MBG, in the pathogenesis
of some forms of hypertension, including preE [3,4,16]. Among the numerous factors contributing to
the development of preE is the significant role played by the CTS and the imbalance of angiogenic
and antiangiogenic factors [17]. There are two types of CTS: OUB and bufadienolides [MBG and
Cinobufotalin (CINO)]. MBG may be secreted in response to the stimulus of excessive volume
expansion in the first trimester in sensitive pregnant patients. It has been shown that urinary MBG
levels are elevated prior to the development of hypertension, indicating that it may play a key role in
the pathogenesis of preE. CTS are stimulated to promote natriuresis via inhibition of the Na+/K+ pump
in renal tubules and are likely to exhibit a prohypertensiveaction via inhibition of the Na+/K+ pump
in vascular sarcolemma. CTS inhibit Na+/K+ ATPase in vascular smooth muscle cells; the elevation of
local Na+ facilitates Ca2+ entry through NCX1, resulting in vasoconstriction [18].
MBG levels are elevated in preE. PreE is a syndrome which is generally believed to involve
multiple etiologic factors. Current research focuses mainly on three pathogenetic mechanisms;
(1) over-secretion of MBG, (2) an imbalance of pro- and anti-angiogenic factors, and (3) agonistic
autoantibodies to the angiotensin II type 1 receptor [
of MBG in preE. CTS ‘endogenous digoxin-like factors’ (EDLF) [11] have been known since the 1980s
to increase significantly during pregnancy-induced hypertension and preE [19 21]. Recently, MBG was
3]. Recent data strongly support the involvement
–
identified as the EDLF. MBG, but not EO, is markedly elevated in PE [22]. Puschett and co-workers
developed an ELISA with high specificity for MBG [23], which revealed a five-fold increase in serum
MBG and a four-fold increase in urine MBG levels in preeclamptic patients versus normotensive
pregnant women [24]. Plasma levels of MBG, but not EO, become elevated in patients with moderate
PE [25]. In addition to the effects of MBG on Na+K+ ATPase and fibrotic signaling, MBG has also been
shown to interfere with the proliferation, migration, and invasion of cytotrophoblast (CTB) cells [26
–28]
and to disrupt endothelial cell junctions [29 30]. Recently, we demonstrated that CINO impedes Sw.71
,
CTB cell line function via cell cycle arrest and apoptotic signaling [31].
Disruption of angiogenic balance by MBG is hypothesized to contribute to preE [32–34].
Angiogenic imbalance during pregnancy may contribute to preE [35–37]. When administered to
pregnant rats, endogenous sFlt-1 and sEng induce several features of preE, including hypertension,
proteinuria, and HELLP syndrome [38]. PlGF is expressed in the placenta and is proangiogenic [39].
sFlt-1 inhibits PlGF. In preE patients, both sFlt-1 and sEng levels are increased prior to clinical symptoms
and also correlate with the severity of the disease, whereas PlGF levels are significantly decreased [40].
The sFlt-1/PlGF ratio, while not yet FDA approved, appears to have diagnostic and predictive value in
patients at risk of developing PE [40], and tests like the CobasElecsys® (Rotkreuz, Switzerland) for preE

Int. J. Environ. Res. Public Health 2017, 14, 517
3 of 15
(sFlt-1 & PlGF) (Roche) are increasingly used in the clinical management of PE [41]. MBG has been
shown to decrease expression of pro-angiogenic factors VEGF and PlGF, while up-regulating expression
of anti-angiogenic factors sFlt-1 and sEng in a cell line of extravillous CTBs. These effects were seen
between 0.1 nM and 1 nM for MBG, which is within the physiological range 0.25 + 0.02 nM [22].
Antibodies to CTS decrease the blood pressure in preE. Additional evidence for the role of CTS in preE
comes from studies in which intravenously administered Digibind lowered blood pressure in patients
with PE [20,21]. It has been suggested that MBG induces vascular fibrosis via a Fli-1-dependent
mechanism, thus suggesting that MBG represents a potential target for therapy of preE [42].
Synthetic receptors (SR) are architecturally designed by chemists for selective recognition of a variety
of substrates, mimicking many critical enzymatic activities involved in biology [43–51]. The unique
functional groups present in such molecules can be potentially used for designing drugs to target specific
activities in a human body [45]. Recently, we used a synthetic receptor on CTB cells, demonstrating
that it impaired the function of human first trimester CTB cells [52]. In order to better understand the
cell surface receptors for CTS and their interactions with synthetic receptors, we studied four acyclic
compounds (Figure 1): Para Nitro Tripodal Urea (PNTU), Penta Fluoro Tripodal Urea (PFTU), Para Nitro
Tripodal Thio-Urea (PNTTU), and Penta Fluoro Tripodal Thio-Urea (PFTTU) and three cyclic compounds
(Figure 2): Thiophene-based Ethylene Macrocyclic Amine (TEA), Copper(II) complex of Methylated
Para-xylyl-based Ethylene Macrocyclic Amine (CuMEPEA), and Macrocyclic Amide (MACHAM).
As shown in Figure 1, the basic feature of these compounds is that each of them contains three active
functional groups as urea [HN(C=O)NH] groups (PNTU and PFTU) or thiourea [HN(C=S)NH] groups
(PNTTU and PFTTU) in the attached three arms forming a triopdal cavity. In addition, the presence of
electron-withdrawing groups on the aromatic units enhances the acidity of the attached NH groups,
thereby increasing the overall activity of these molecules. In addition, the conformational flexibility with
six H-donor groups may allow them to interact with cells. In order to understand the effectiveness of
such compounds, we included three macrocyclic-based compounds, as shown in Figure 2, featuring
amine (TEA), metal center (CuMEPEA), and amide (MACHAM) functional groups.
NO2
NO2
S
O
N
H
N
H
N
N
H
H
H
H
H
H
N
NO2
N
NO2
N
N
N
N
O
H
N
S
H
H
N
N
H
N
O
S
NO2
NO2
PNTTU
PNTU
F
F
F
F
F
F
S
O
N
H
N
H
F
F
F
F
N
N
H
H
F
F
F
F
F
H
H
N
H
H
N
N
F
N
F
N
N
F
F
F
O
H
N
H
F
F
S
F
F
H
N
N
H
N
O
S
F
F
F
F
F
F
PFTU
PFTTU
Figure 1. Chemical structures of acyclic receptors: PNTU (Para Nitro Tripodal Urea), PFTU (Penta
Fluoro Tripodal Urea), PNTTU (Para Nitro Tripodal Thio-Urea), and PFTTU (Penta Fluoro Tripodal
Thio-Urea).

Int. J. Environ. Res. Public Health 2017, 14, 517
4 of 15
S
NH
N
N
Cu2+
N
HN
HN
Br-
Cu2+
Br-
-Br
-Br
NH
N
N
HN
N
NH
S
CuMEPEA
TEA
O
NH
NH
O
N
N
O
N
HN
N
H
N
N
N
O
N
N
O
NH
NH
O
MACHAM
Figure 2. Chemical structures of cyclic receptors: TEA (Thiophene-based Ethylene Macrocyclic Amine),
CuMEPEA (Copper(II) complex of Methylated Para-xylyl-based Ethylene Macrocyclic Amine) and
MACHAM (Macrocyclic Amide).
While preE is considered to be a multifactorial disease, a consensus among preE researchers has
emerged that MBG levels are important in at least some cases [32]. MBG levels are elevated in most PE
patients. In vitro data demonstrate the effects of MBG on endothelial function [30] and cell growth [53
]
and, importantly, on proliferative and angiogenic signalling in CTB cells [26 28]. Compelling data
–
from multiple animal models, in which MBG levels are elevated prior to the onset of preE, suggests
that MBG may represent a relevant target for preE therapeutics. Indeed, the administration of DigiBind
was effective at alleviating hypertension in preE patients. Anti-MBG antibodies have alleviated preE
symptoms in a rodent model. We have identified a novel anti-MBG human monoclonal antibody, which
binds with high affinity to NKA and attenuates MBG-induced anti-proliferative and anti-angiogenic
signaling in a CTB cell model [34], as an innovative, effective, and safe therapeutic for preE.
In the present study, we investigated the seven selected synthetic receptors with different function
groups to explore their effectiveness on the angiogenic profile of human first trimester CTB cells.
2. Materials and Methods
2.1. General Information
The chemicals used for this work were purchased from Sigma-Aldrich as reage◦nt grades and
were used as received. Nuclear magnetic resonance (NMR) spectra were recorded at 25 C on a Varian
Unity INOVA 500 FT-NMR. Chemical shifts for NMR were expressed in parts per million (ppm) and
calibrated against trimethylsilane (TMS) or sodium salt of 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid
(TSP) as an external reference used in a sealed capillary tube. All NMR data were processed and
analyzed with MestReNova Version 6.1.1-6384 (Mestrelab, Escondido, CA, USA). Mass spectral data
were obtained at ESI-MS positive mode on a FINNIGAN LCQDUO (Jackson State University, Jackson,
MS, USA). Elemental analysis was done from Columbia Analytical Service (Tucson, AZ, USA). The CTB
cell culture media DMEM/F-12 was purchased from Invitrogen, Grand Island, NY, USA, and the cells
were incubated in an Isotemp CO2 Incubator, Fisher, Waltham, MA, USA. RPMI Media and gels were

Int. J. Environ. Res. Public Health 2017, 14, 517
5 of 15
purchased from Invitrogen, Grand Island, NY, USA. Cell viability and cell proliferation assay kits were
purchased from Promega, Madison, WI, USA. A cell migration assay kit was purchased from Cell
Biolabs, San Diego, CA, USA. The BCA protein assay kit and chemiluminescent substrate were from
Pierce, Rockford, IL, USA. The Quantikine ELISA was purchased from R&D Systems, Minneapolis,
MN, USA. The nitrocellulose membranes were from Bio-Rad, Hercules, CA, USA. The primary and
secondary antibodies were purchased from Santa Cruz Biotechnology, Paso Robles, CA, USA; Abcam,
Cambridge, MA, USA and Jackson Immuno Research Laboratories, West Grove, PA, USA. Absorbance
was measured on a plate reader, SPECTRA max 340PC384, Molecular Devices, Sunnyvale, CA, USA.
The fluorescence was measured on a fluorescence plate reader, CytoFluor Series 4000 Fluorescence
Multi-Well Plate Reader, Applied Biosystems, Grand Island, NY, USA. The chemiluminence detection
system used was the LAS-3000 Imaging System, Fuji Photo Film Co., Ltd., Minato-ku, Tokyo, Japan.
2.2. Synthesis
Para Nitro Tripodal Urea (PNTU): Tris(2-aminoethyl)amine (0.33 g, 2.26 mmol) and p-nitrophenyl
isocyanate (1 g, 6.09 mmol) were added in dichloromethane (250 mL). The reaction mixture was
refluxed for 6 h. A yellowish precipitate was formed and collected by filtration followed by washing
1
with CH2Cl2 to get desired product. Yield: 1.26 g, 95%. H NMR (500 MHz, DMSO-d6, TSP):
δ
9.32
(s, 3H, Ar-NH), 8.08 (d, J = 9.10 Hz, 6H, ArH), 7.58 (d, J = 8.6 Hz, 6H, ArH), 6.41 (t, J = 5.75 Hz 3H,
CH2NH), 3.22 (m, J = 6.15 Hz, 6H, NHCH2), 2.63 (t, J = 6.65 Hz, 6H, NCH2). 13C NMR (125 MHz,
DMSO-d6):
δ 176.64 (C=O), 154.8 (Ar-C), 147.5 (ArC-NO2), 140.8 (Ar-CH), 125.4 (Ar-CH), 54.1 (NHCH2),
37.8 (NCH2). ESI-MS: m/z (%) 639.23 [M + H]+. Anal. Calcd. for C27H30N10O6S3: C, 50.78; H, 4.74; N,
21.93. Found: C, 50.77; H, 4.75; N, 21.95.
Penta Fluoro Tripodal Urea (PFTU): A CH2Cl2 solution (200 mL) of pentafluorophenyl isocyanate
(2.62 mL, 20.04 mmol) was added to tris(2-aminoethyl) amine (1 mL, 6.69 mmol) solution (100 mL
CH2Cl2) with constant stirring at room temperature. The reaction mixture was refluxed for 4 h to
get a white precipitate, which was collected by filtration. Yield: 4.65 g, 90%. 1H NMR (500 MHz,
DMSO-d6, TSP):
δ
8.32 (s, 3H, Ar-NH), 6.53 (t, J = 5.5 Hz, 3H, CH2NH), 3.15 (m, J = 6.6 Hz, 6H, NHCH2),
154.8 (C=O), 144.4 (Ar-C), 139.4
2.57 (t, J = 6.65 Hz, 6H, NCH2). 13C NMR (125 MHz, DMSO-d6):
δ
(ArC-F), 136.9 (ArC-F), 115.2 (ArC-F), 54.07 (NHCH2), 38.4 (NCH2). ESI-MS: m/z (%) 774.23 [M + H]+.
Anal. Calcd. for C27H18F15N7O0: C, 41.93; H, 2.35; N, 36.84. Found: C, 42.95; H, 2.33; N, 36.86.
Para Nitro Tripodal Thio-Urea (PNTTU): This compound was prepared following the procedure
as descried earlier [52].
Penta Fluoro Tripodal Thio-Urea (PFTTU): A solution of pentafluorophenyl isothiocyanate
(586.3
tris(2-aminoethyl) amine (200
0.5 h. The reaction mixture was refluxed for 36 h, yielding a white precipitate. Yield: 0.96 g, 87%.
1H NMR (500 MHz, DMSO-d6, TSP):
9.30 (s, 3H, Ar-NH), 8.08 (s, 3H, CH2NH), 3.57 (t, J = 5.15 Hz, 6H,
NHCH2), 2.73 (t, J = 6.07 Hz, 6H, NCH2). 13C NMR (125 MHz, DMSO-d6):
182.3 (C=S), 144.9 (Ar-C),
µL, 4.15 mmol) dissolved in dichloromethane (50 mL) was added to a solution of
µL, 1.34 mmol) dissolved in dichloromethane (50 mL) slowly over
δ
δ
142.9 (ArC-F), 140.5 (ArC-F), 138.2 (ArC-F), 136.2 (ArC-F), 114.9 (ArC-F), 51.9 (NHCH2), 42.7 (NCH2).
M.P 190 ◦C. ESI-MS: m/z (%) 822.65 [M + H]+. Anal. Calcd. for C27H18F15N7S3: C, 39.47; H, 2.21; N,
11.93. Found: C, 39.45; H, 2.23; N, 11.97.
Thiophene-based Ethylene Macrocyclic Amine (TEA): Diethylenetriamine (1.00 g, 9.70 mmol)
and 2,5-thiophenedicarboxaldehyde (1.35 g, 9.70 mmol) were reacted at room temperature under high
1
dilution condensation in MeOH (400 mL), followed by NaBH4 reduction. Yield: 1.23 g, 65%. H NMR
(300 MHz, CDCl3, TMS): δ 6.73 (s, 4H, ArH), 3.87 (s, 8H, ArCH2), 3.77 (t, 8H, CH2), 2.72 (t, 8H, CH2)
Anal. Calcd. for C20H34N6S2: C, 56.8; H, 8.1; N, 19.9. Found: C, 56.6; H, 8.2; N, 20.1.
Copper(II) complex of Methylated Para-xylyl-based Ethylene Macrocyclic Amine (CuMEPEA):
The free macrocycle (MEPEA) was prepared by a high dilution condensation reaction of
tera-phthalaldehyde (0.01 mol) and N-methyl-2, 20-diaminodiethylamine (0.01 mol) in methanol
(400 mL) followed by NaBH4 reduction. Yield: 1.09 g, 43%. M.p. 88 ◦C. 1H NMR (500 MHz, CDCl3,

Int. J. Environ. Res. Public Health 2017, 14, 517
6 of 15
TMS):
δ
7.19 (s, 8H, ArH), 3.75 (s, 8H, ArCH2), 2.78 (t, J = 5.0 Hz, 8H, NHCH2), 2.54 (t, J = 5.0 Hz
138.91 (Ar-C), 127.93 (Ar-C),
8H, NHCH2CH2), 2.16 (s, 6H, NCH3). 13C NMR (125 MHz, CDCl3):
δ
56.50 (CH3NCH2), 53.93 (ArCH2), 46.95 (NCH2CH2) 42.14 (NCH3). ESI-MS: m/z (+) 439.3 [M + H]+.
Anal. Calcd. for C26H42N6: C, 71.19; H, 9.65; N, 19.16. Found: C, 71.33; H, 9.67; N, 19.21. MEPEA
(4.0 mmol) dissolved in MeOH (5 mL) and CuBr2 (8.0 mmol)dissolved in H2O (5 mL) were mixed in
MeOH (5 mL). The solution was stirred continuously, and the blue solution was heated to 60 ◦C for
2 min under stirring. The solution was then kept for crystallization. After ten days, deep blue crystals
were collected by fi◦ltration, washed by diethyl ether and wiped out with tissue. Yield 167.2 mg, 70%
yield, Mp: 214–215 C (decom). Anal. Calcd. For C26H44Br4Cu2N6O: C, 34.57; H, 4.91; N, 9.30. Found:
C, 34.61; H, 4.90; N, 9.33.
Macrocyclic Amide (MACHAM): The free macrobicycle (1.0 gm, 1.66 mmol) was added to
p-cyanophenyl isocyanate (1.144 g, 10 mmol) in 250 mL of CH2Cl2 at room temperature. The reaction
mixture was refluxed for 24 h. A white precipitate was formed and collected by filtration, followed
1
by washing with diethyl ether. Yield 90%. H NMR (500 MHz, DMSO-d6):
δ 9.19 (s, 6H, CO-NH),
7.73 (d, 12H, J = 14.6 Hz, ArH), δ 7.64 (d, 12H, J = 14.6 Hz, ArH), 7.01 (s, 12H, ArH), 4.39 (s, 12H, ArCH2),
3.16 (m, 12H, NCH2), 1.61 (m, 12H, NCH2). 13C NMR (125 MHz, DMSO-d6):
δ
155.2 (C=O), 146.1
(Ar-CN), 138.6 (Ar-C), 133.7 (Ar-CH), 129.7 (Ar-CH), 120.3 (Ar-CH), 119.9 (Ar-CH), 119.2 (Ar-C), 103.8
(Ar-CH), 55.7 (NCH2), 51.7 (NCH2). ESI-MS: m/z 1485.65 [M + Na]+. Anal. Calcd. for C84H78N20O6: C,
68.93; H, 5.37; N, 19.14. Found: C, 68.95; H, 5.38; N, 19.35.
2.3. Cell Culture
The human extravillous CTB cell line Sw.71 utilized in these studies was derived from first
trimester chorionic villus tissue and was kindly provided by Gil G. Mor at Yale University School of
Medicine, New Haven, CT, USA. These cells are well characterized and share many characteristics with
isolated primary cells, including the expression of cytokeratin-7, human leukocyte antigen (HLA) class
I antigen, HLA-G, BC-1, CDp, human chorionic gonadotropin, and human placental lactogen [1–4].
Sw.71 cells were cultured in DMEM/F-12 (Invitrogen) supplemented with 10% fetal bovine serum,
10 mM Hepes, 0.1 mM MEM non-essential amino acids, 1 mM sodium pyruvate, and 100 U/mL
penicillin/streptomycin. Cells were incubated at 37 ◦C, 5% CO2, and 99% humidity (Fisher, Isotemp
CO2 Incubator).
2.4. Treatment of Cells
Cells were treated with RPMI Media (Invitrogen) containing 0.1, 1, 10, or 100 nM SR for 48 h.
The supernatants were collected to measure the levels of angiogenic and anti-angiogenic factors
using ELISA.
2.5. ELISA for Angiogenic and Anti-Angiogenic Factors
The supernatants of the TRSPHNO2 treated cells were analyzed using a Quantikine ELISA
(R&D Systems) for concentrations of sVEGF R1/Flt-1 (sFlt-1), and VEGF. These assays employ the
quantitative sandwich enzyme immunoassay technique. Monoclonal antibodies specific for sFlt-1 and
VEGF were pre-coated onto the microplate. Standards and samples are pipetted into the wells and any
sFlt-1 and VEGF present is bound by the immobilized antibody. After washing away any unbound
substances, an enzyme-linked polyclonal antibody specific for either sFlt-1 and VEGF is added to the
wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is
added to the wells, and color develops in proportion to the amount of sFlt-1 and VEGF bound in the
initial step. The color development is stopped and the intensity of the color is measured using a plate
reader (SPECTRAmax 340PC384).

Int. J. Environ. Res. Public Health 2017, 14, 517
7 of 15
2.6. Western Blots for VEGFR-1, AT1, and AT2 Receptors
Cells were treated with RPMI Media (Invitrogen) containing 0.1, 1, 10, or 100 nM SR for 48 h.
The cell lysates were utilized to measure VEGFR-1, AT1, and AT2 receptor expression. Protein
concentrations of cell lysates were determined using a BCA Protein Assay Kit (Pierce). An equal
amount of protein from each sample was run on a NuPAGENovex 4–12% Bis-Tris Gel (Invitrogen) and
transferred to 0.45 µm nitrocellulose membrane (Bio-Rad). Membranes were blocked in 5% milk and
incubated with VEGFR-1 (Invitrogen), AT1, (Santa Cruz Biotechnology), and AT2 (Abcam) antibodies.
After the addition of the corresponding secondary antibody (Jackson ImmunoResearch Laboratories),
proteins were visualized with SuperSignal West Dura Chemiluminescent Substrate (Pierce) and a
chemiluminescence detection system (LAS-3000 Imaging System). Densitometry was measured using
ImageJ software (National Institutes of Health, Bethesda, MA, USA,) and normalized using
(ABCAM, Cambridge, MA, USA).
β-actin
2.7. Statistical Method
Data are presented as mean
(DMSO)-treated groups using a one-way analysis of variance with Tukey’s post hoc test. A p-value of
±
SEM. Data from CINO-treated groups were compared to basal
less than 0.05 was considered significant.
3. Results
3.1. PNTU, PFTU, PNTTU, PFTTU, and CuMEPEA Downregulated Angiogenic Factors
The secretion of VEGF was inhibited in the culture media of CTB cells treated with
≥
1 nM of
PNTU, PFTU, PNTTU, PFTTU, and CuMEPEA while the angiogenic factors in the culture media of
CTB cells had no effect when they were treated with 0.1 nM of those compounds. TEA Cycle and
MACHAM had no effect on VEGF secretion in CTB cells (Figure 3). These results suggested that the
SR had an anti-angiogenic effect on CTB cells environment.
900
800
700
PNTU
600
500
400
300
200
100
0
PFTU
*
*
*
*
*
*
*
*
*
PNTTU
*
*
*
*
*
*
PFTTU
TEA Cycle
CUMEPEA
MACHAM
Basal
0.1 nM
1 nM
10 nM
100 nM
Figure 3. Cytotrophoblast (CTB) cells were treated with different concentrations of synthetic receptors
(SRs), and the levels of Vascular Endothelial Growth Factor (VEGF) were measured in the cell
culture media by Enzyme-Linked Immunosorbent Assay (ELISA). The SRs significantly (* p < 0.05)
downregulated the secretion of VEGF by CTB cells. The results are presented as the mean
Error Mean (SEM) (n = 6, 4 replicates each).
±
Standard
3.2. PNTU, PFTU, PNTTU, PFTTU Upregulated Anti-Angiogenic Factors
The secretion of sFlt-1 was significantly upregulated in the culture media of CTB cells treated with
1 nM PNTU, PFTU, PNTTU, and PFTTU, while the secretion of sFlt-1 did not have much effect on
the culture media of CTB cells treated with 0.1 nM of PNTU, PFTU, PNTTU, and PFTTU. TEA Cycle
≥

Int. J. Environ. Res. Public Health 2017, 14, 517
8 of 15
and MACHAM had no effect on sFlt-1 secretion. (Figure 4) Therefore, the SRs have demonstrated an
anti-angiogenic effect on the CTB cells.
160
*
*
140
120
100
80
*
*
*
*
*
*
*
*
*
*
*
*
*
PNTU
PFTU
PNTTU
PFTTU
TEA Cycle
CUMEPEA
MACHAM
60
40
20
0
Basal
0.1 nM
1 nM
10 nM
100 nM
Figure 4. CTB cells were treated with different concentrations of SRs, and the levels of sFlt-1 were
measured in the cell culture media by ELISA. The SRs significantly (* p < 0.05) upregulated the secretion
of sFlt-1 by CTB cells. The results are presented as the mean ± SEM (n = 6, 4 replicates each).
3.3. PNTU, PFTU, PNTTU, PFTTU, CuMEPEA, TEA Cycle and MACHAM Upregulated AT2 Receptor
Expression
The AT2 receptor expression was significantly upregulated in the culture media of CTB cells
treated with
≥
1 nM PNTU, PFTU, PNTTU, PFTTU, CuMEPEA, TEA cycle, and MACHAM compared
to basal (Figure 5). There is a possibility that AT2 receptor mediated vasodilation plays a role in
modulating Ang-II contractile responses in pregnancy.
Figure 5. CTB cells were treated with different concentrations of SRs, and Angiotensin II receptor, type
2 (AT2) receptor expression was measured in the cell lysates by Western Blot. The SRs significantly
(
* p < 0.05) upregulated the expression of AT2 receptor in CTB cells. The data are presented as mean
±
SEM for four experiments. One blot from each of the seven SRs is shown.

Int. J. Environ. Res. Public Health 2017, 14, 517
9 of 15
3.4. PNTU, PFTU, PNTTU, PFTTU, CuMEPEA, TEA and MACHAM Downregulated VEGFR-1 and AT1
Receptor Expression
The VEGFR-1 and AT1 receptor expression was downregulated in the culture media of CTB cells
treated with
≥
1 nM PNTU, PFTU, PNTTU, PFTTU, CuMEPEA, TEA cycle, and MACHAM, compared
to basal (Figure 6). However studies have demonstrated that the activation of AT1 receptor appears to
mediate hypertension associated with excessive IL-6 during preE.
1.1
1
*
*
*
PNTU
0.9
0.8
0.7
0.6
0.5
*
*
PFTU
*
*
*
*
*
*
*
PNTTU
*
*
*
*
*
*
PFTTU
*
*
*
TEA Cycle
CuMEPEA
MACHAM
*
*
* *
*
*
*
Basal
0.1 nM
1 nM
10 nM
100 nM
Figure 6. CTB cells were treated with different concentrations of SRs, and Vascular endothelial growth
factor receptor 1 (VEGFR1) receptor expression was measured in the cell lysates by Western Blot.
The SRs significantly (* p < 0.05) downregulated the expression of VEGFR1 receptor in CTB cells.
The data are presented as mean
is shown.
±
SEM for four experiments. One blot from each of the seven SRs
3.5. PNTU, PFTU, PNTTU, PFTTU, and CuMEPEA Downregulated AT1
The expression of AT1 receptor expression was significantly downregulated for CTB cells treated
with 0.1 and 1 nM PNTU, PFTU, PNTTU, PFTTU, and CuMEPEA; however AT1 receptor expression
was not changed in CTB cells treated with 10 and 100 nM of those compounds (Figure 7).

Int. J. Environ. Res. Public Health 2017, 14, 517
10 of 15
1.1
1
*
*
*
*
PNTU
*
*
*
*
*
*
*
PFTU
*
0.9
0.8
0.7
0.6
*
*
*
*
*
PNTTU
*
*
*
*
*
*
*
PFTTU
*
TEA Cycle
CuMEPEA
MACHAM
*
Basal
0.1 nM
1 nM
10 nM
100 nM
Figure 7. CTB cells were treated with different concentrations of SRs, and Angiotensin II receptor, type
1 (AT1) receptor expression was measured in the cell lysates by Western Blot. The SRs significantly
(* p < 0.05) downregulated the expression of AT1 receptor in CTB cells. The data are presented as mean
± SEM for four experiments. One blot from each of the seven SRs is shown.
4. Discussion
As compared to preE pregnancies, normal pregnancies reveal a higher ratio of angiogenic
factors to anti-angiogenic factors that then ensures the human extra villous CTB cells to proliferate,
migrate, and invade the chorionic villus tissue for the establishment and growth of a healthy placenta.
Two angiogenic factors that are vital for normal placental progression are VEGF and PlGF. Within
an individual who has developed preE, the anti-angiogenic factors are produced at elevated levels.
Through varying data and tests that we have run, such as the ELISA and the Western Blot [27],
we propose that cardiotonic steroids being secreted throughout the body may cause this imbalance.
The CTS inhibited CTB proliferation, migration, invasion, and ERK1/2 phosphorylation, and they
activated Jnk1/2 phosphorylation, p38 phosphorylation, and apoptosis, as evaluated by caspase 3/7
and annexin-five staining [27
,28]. The CTS also arrested cell cycle progression without causing a
cytotoxic effect on the cells [27
,28]. We have demonstrated that CTS-induced impairment of CTB
cell function occurs via the modulation of MAPK signaling, cell cycle arrest, and the activation of
apoptosis. However, the cell surface receptors for CTS have been poorly understood. CINO impairs
CTB cell function via cell cycle arrest and apoptotic signaling [32]. Since mechanistic insights of the
release of the cardiotonic steroids are still not well known, the proper selection of effective functional
groups of synthetic compounds may elucidate the location of cardiotonic binding to the CTB cells.
This can be accomplished by the binding of multi-functional SRs to MBG, which enables the tracking
of MBG to the binding sites. In the long run, this may help us find a treatment to help prohibit the
premature apoptosis signaling in CTB cells. In order to better understand the cell surface receptors
for CTS, we decided to test seven selected new compounds; namely, (PNTU), (PFTU), (PNTTU),
(PFTTU), TEA, CuMEPEA, and MACHAM. When looking at the CTB cells treated with
≥
1 nM of

Int. J. Environ. Res. Public Health 2017, 14, 517
11 of 15
PNTU, PFTU, PNTTU, PFTTU, and CuMEPEA, the secretions of VEGF and PlGF were decreased.
However, TEA and MACHAM had no effect on VEGF secretion. When observing anti-angiogenic
regulation, the secretion of sFlt-1 and sEnd were increased in the CTB cells treated with
≥
1 nM
PNTU, PFTU, PNTTU, PFTTU, and CuMEPEA. However, TEA and MACHAM had no effect on sFlt-1
secretion. It is proposed that the urea/thiourea [HN(C=O/S) NH] groups in PNTU, PFTU, PNTTU,
and PFTTU possibly interact with the active sites of cells through hydrogen bonding interactions.
In addition, the presence of three electron-withdrawing substituents (as p-nitro groups or penta fluoro
groups) enhances the acidity of the attached NH groups, thereby increasing the overall activity of
the ligand, as well as the conformational flexibility with six H-donor groups, which may allow the
ligand to interact with cells more comfortably [51]. The receptor CuMEPEA also interacts with the
cells, possibly due to the presence of the active Lewis acid centers as copper(II) within the receptor’s
framework. In contrast, TEA and MACHAM, which contain different functional groups, amines,
and amides, respectively, exhibited poor interactions with the cells under the experimental conditions.
These observations suggest that the poor acidity of the two functional groups diminishes the ability
of these compounds to interact with the cells under neutral conditions [43
expression was significantly upregulated; however, the AT1 and VEGFR-1 receptor expression was
significantly downregulated in CTB cells treated with with 1 nM PNTU, PFTU, PNTTU, PFTTU,
,44]. The AT2 receptor
≥
and CuMEPEA. However, TEA and MACHAM showed negligible interactions due to the presence
of amine and amide functionalities, which are less acidic than urea and thiourea functionalities
under neutral conditions [44]. AT2 receptor is antiangiogenic and antiproliferative; however, AT1
and VEGFR1 receptors are angiogenic [54]. AT1 and AT2 have discrete physiological actions from
each other; activation of AT1 induces vasoconstriction and cell proliferation, while AT2 facilitates
vasodilatation, hinders cell growth and triggers apoptosis [55]. Thus these data confirm our data on
VEGF and s-Flt-1 as well as our previous data for CTS and SR [28,29,33,52,56].
PreE is a hypertensive disorder that develops in three to 10 percent of pregnant women during
their first trimester of gestation [57,58]. In the first trimester of pregnancy, the CTB cells of the extra
villous trophoblast column migrate and invade the decidualized endometrium attaching the placenta
to the uterus. The CTB cells subsequently breach and line the uterine blood vessels, leading to the
channeling of the maternal blood to the placenta. Adequate CTB invasion leads to the remodeling of
the maternal vessels, which is essential for the fetus, which needs increased maternal blood flow as the
pregnancy progresses. Defects in CTB differentiation are often associated with preE since there will
be a reduction in the uteroplacental perfusion that will further lead to placental focal ischemia and
hypoxia later in pregnancy.
In addition to inhibiting CTB proliferation, migration, and invasion, MBG has been shown to
increase phosphorylated p38 concentration in CTBs, which is considered to be the active protein form
that initiates a stress signaling cascade [27,28]. It has been shown that hypoxic stress activates p38
signaling in villous trophoblasts in preE patients [37]. COX-2, a prostaglandin endoperoxide synthase,
catalyzes the conversion of prostaglandin from arachidonic acid and is considered to be at an increased
level in the early trophoblast tissue [35
–37]. It has been shown that COX-2 is increased in the placenta
tissue of preE patients [38 39]. However, the exact mechanism by which COX-2 is increased in preE
,
and the interaction between CTSs and COX-2 is not well understood.
5. Conclusions
During this study, we have explored several synthetic receptors by varying the functional
groups, including ureas, thioureas, metal centers, amines, and amides to identify their effectiveness
on an angiogenic profile of CTB cells. In particular, we have shown that the receptors containing
urea/thiourea groups are highly effective at downregulating CTB proliferation without any cytotoxic
effect on the CTB cells. This has been proven by a cell viability test. Our results demonstrated that the
synthetic receptors significantly decreased CTB cell proliferation at 100 nM, whereas cell viability at
100 nM remains the same as at basal and lower concentrations [33,34,56]. The molecules containing

Int. J. Environ. Res. Public Health 2017, 14, 517
12 of 15
active binding sites and defined cavities are known to provide strong affinity for anionic species
through H-bonding and electrostatic interactions under neutral conditions [44]. We propose that the
specific binding sites within the synthetic receptors possibly interact with membrane receptors through
residual anionic carboxyl groups, resulting in the decrease in signaling for an Angiogenic profile.
The results obtained from this study will be useful to design an effective inhibitor to prevent the
CTS-induced impairment of CTBs. Taken together, the results from our study will serve as a prototype
for the rational design of highly efficient synthetic receptors for biomedical applications.
Acknowledgments: Funding for this work was provided by the Scott, Sherwood and Brindley Foundation,
the Department of Obstetrics and Gynecology (Mohammad Nasir Uddin), the Noble Centennial Endowment
for Research in Obstetrics and Gynecology (Thomas J. Kuehl), and Scott & White Healthcare, Temple, TX, USA.
The CTB cell line Sw.71 was kindly provided by Gil G. Mor at the School of Medicine, Yale University, New Haven,
CT, USA. The project described was supported by Grant Number G12MD007581 from the National Institutes of
Health. Medical Writer Adam Stephens (Scott & White Healthcare, Temple, TX, USA) assisted in editing and
formatting the manuscript.
Author Contributions: Ahmed F. Pantho, Mason Price, AHM Zuberi Ashraf, Umaima Wajid, Afsana Jahan,
and Syeda H. Afroze performed the experiments and drafted and revised the final manuscript as submitted.
Md. Alamgir Hossain, Afsana Jahan, Maryam Emami Khansari, Md Mhahabubur Rhaman, and Corey
R. Johnson designed and conceptualized the synthesis of the synthetic compound, critically reviewed the
manuscript, and approved the final manuscript as submitted. Thomas J. Kuehl performed the statistical analysis,
critically reviewed the manuscript, and approved the final manuscript as submitted. Mohammad Nasir Uddin
conceptualized and designed the study, carried out data interpretation, revised the article critically, and approved
the final manuscript as submitted.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
Von Dadelszen, P.; Magee, L.A. Pre-eclampsia: An update. Curr. Hypertens. Rep. 2014, 16, 454. [CrossRef]
[PubMed]
Steegers, E.A.; Von Dadelszen, P.; Duvekot, J.J.; Pijnenborg, R. Pre-eclampsia. Lancet 2010, 376, 631–644.
[CrossRef]
Puschett, J.B.; Agunanne, E.; Uddin, M.N. Emerging role of the bufadienolides in cardiovascular and kidney
diseases. Am. J. Kidney Dis. 2010, 56, 359–370. [CrossRef] [PubMed]
Uddin, M.N.; Allen, S.R.; Jones, R.O.; Zawieja, D.C.; Kuehl, T.J. Pathogenesis of pre-eclampsia:
Marinobufagenin and angiogenic imbalance as biomarkers of the syndrome. Transl. Res. 2012, 160, 99–113.
[CrossRef] [PubMed]
5.
6.
7.
Khan, K.S.; Wojdyla, D.; Say, L.; Gulmezoglu, A.M.; Van Look, P.F. WHO analysis of causes of maternal
death: A systematic review. Lancet 2006, 367, 1066–1074. [CrossRef]
Duley, L. The global impact of pre-eclampsia and eclampsia. Semin. Perinatol. 2009, 33, 130–137. [CrossRef]
[PubMed]
Berg, C.J.; Mackay, A.P.; Qin, C.; Callaghan, W.M. Overview of maternal morbidity during hospitalization
for labor and delivery in the United States: 1993–1997 and 2001–2005. Obstet. Gynecol. 2009, 113, 1075–1081.
[CrossRef] [PubMed]
8.
9.
Wallis, A.B.; Saftlas, A.F.; Hsia, J.; Atrash, H.K. Secular trends in the rates of preeclampsia, eclampsia, and
gestational hypertension, United States, 1987–2004. Am. J. Hypertens. 2008, 21, 521–526. [CrossRef] [PubMed]
Lin, S.; Leonard, D.; Co, M.A.; Mukhopadhyay, D.; Giri, B.; Perger, L.; Beeram, M.R.; Kuehl, T.J.; Uddin, M.N.
Pre-eclampsia has an adverse impact on maternal and fetal health. Transl. Res. 2015, 165, 449–463. [CrossRef]
[PubMed]
10. Bagrov, A.Y.; Shapiro, J.I.; Fedorova, O.V. Endogenous cardiotonic steroids: Physiology, pharmacology, and
novel therapeutic targets. Pharmacol. Rev. 2009, 61, 9–38. [CrossRef] [PubMed]
11. Buckalew, V.M. Endogenous digitalis-like factors: An overview of the history. Front. Endocrinol. 2015, 6, 49.
[CrossRef] [PubMed]
12. Fedorova, O.V.; Agalakova, N.I.; Morrell, C.H.; Lakatta, E.G.; Bagrov, A.Y. ANP differentially modulates
marinobufagenin-induced sodium pump inhibition in kidney and aorta. Hypertension 2006, 48, 1160–1168.
[CrossRef] [PubMed]

Int. J. Environ. Res. Public Health 2017, 14, 517
13 of 15
13. Schoner, W.; Scheiner-Bobis, G. Endogenous and exogenous cardiac glycosides: Their roles in hypertension,
salt metabolism, and cell growth. Am. J. Physiol. Cell. Physiol. 2007, 293, 509–536. [CrossRef] [PubMed]
14. Fedorova, O.V.; Tapilskaya, N.I.; Bzhelyansky, A.M.; Frolova, E.V.; Nikitina, E.R.; Reznik, V.A.; Kashkin, V.A.;
Bagrov, A.Y. Interaction of Digibind with endogenous cardiotonic steroids from preeclamptic placentae.
J. Hypertens. 2010, 28, 361–366. [CrossRef] [PubMed]
15. Liu, J.; Shapiro, J.I. Regulation of sodium pump endocytosis by cardiotonic steroids: Molecular mechanisms
and physiological implications. Pathophysiology 2007, 14, 171–181. [CrossRef] [PubMed]
16. Uddin, M.N.; Agunanne, E.E.; Horvat, D.; Puschett, J.B. Resibufogenin administration prevents oxidative
stress in a rat model of human preeclampsia. Hypertens. Pregnancy 2012, 31, 70–78. [CrossRef] [PubMed]
17. Fedorova, O.V.; Simbirtsev, A.S.; Kolodkin, N.I.; Kotov, A.Y.; Agalakova, N.I.; Kashkin, V.A.; Tapilskaya, N.I.;
Bzhelyansky, A.; Reznik, V.A.; Frolova, E.V.; et al. Monoclonal antibody to an endogenous bufadienolide,
marinobufagenin, reverses preeclampsia-induced Na/K-ATPase inhibition and lowers blood pressure in
NaCl-sensitive hypertension. Hypertension 2008, 24, 14–25. [CrossRef] [PubMed]
18. Iwamoto, T. Vascular Na+/Ca2+ exchanger: Implications for the pathogenesis and therapy of
salt-dependenthypertension. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2006, 290, R536–R545. [CrossRef]
[PubMed]
19. Graves, S.W.; Williams, G.H. An endogenous ouabain-like factor associated with hypertensive pregnant
women. J. Clin. Endocrinol. MeTable 1984, 59, 1070–1074. [CrossRef] [PubMed]
20. Goodlin, R.C. Antidigoxin antibodies in eclampsia. N. Engl. J. Med. 1988, 318, 518–519. [PubMed]
21. Adair, C.D.; Buckalew, V.; Taylor, K.; Ernest, J.M.; Frye, A.H.; Evans, C.; Veille, J.C. Elevated
endoxin-like factor complicating a multifetal second trimester pregnancy: Treatment with digoxin-binding
immunoglobulin. Am. J. Nephrol. 1996, 16, 529–531. [CrossRef] [PubMed]
22. Lopatin, D.A.; Ailamazian, E.K.; Dmitrieva, R.I.; Shpen, V.M.; Fedorova, O.V.; Doris, P.A.; Bagrov, A.Y.
Circulating bufadienolide and cardenolide sodium pump inhibitors in preeclampsia. J. Hypertens. 1999, 17,
1179–1187. [CrossRef] [PubMed]
23. Abi-Ghanem, D.; Lai, X.; Berghman, L.R.; Horvat, D.; Li, J.; Romo, D.; Uddin, M.N.; Kamano, Y.; Nogawa, T.;
Xu, J.P.; et al. A chemifluorescent immunoassay for the determination of marinobufagenin in body fluids.
J. Immunoassay Immunochem. 2011, 32, 31–46. [CrossRef] [PubMed]
24. Agunanne, E.; Horvat, D.; Harrison, R.; Uddin, M.N.; Jones, R.; Kuehl, T.J.; Ghanem, D.A.; Berghman, L.R.;
Lai, X.; Li, J.; et al. Marinobufagenin Levels in Preeclamptic Patients: A Preliminary Report. Am. J. Perinatol.
2011, 28, 509–514. [CrossRef] [PubMed]
25. Averina, I.V.; Tapilskaya, N.I.; Reznik, V.A.; Frolova, E.V.; Fedorova, O.V.; Lakatta, E.G.; Bagrov, A.Y.
Endogenous Na/K-ATPase inhibitors in patients with preeclampsia. Cell. Mol. Biol. 2006, 52, 19–23.
[PubMed]
26. LaMarca, H.L.; Morris, C.A.; Pettit, G.R.; Nagowa, T.; Puschett, J.B. Marinobufagenin impairs first trimester
cytotrophoblast differentiation. Placenta 2006, 27, 984–988. [CrossRef] [PubMed]
27. Uddin, M.N.; Horvat, D.; Glaser, S.S.; Danchuk, S.; Mitchell, B.M.; Sullivan, D.E.; Morris, C.A.; Puschett, J.B.
Marinobufagenin inhibits proliferation and migration of cytotrophoblast and CHO cells. Placenta 2008, 29,
266–273. [CrossRef] [PubMed]
28. Uddin, M.N.; Horvat, D.; Glaser, S.S.; Mitchell, B.M.; Puschett, J.B. Examination of the cellular mechanisms by
which marinobufagenin inhibits cytotrophoblast function. J. Biol. Chem. 2008, 283, 17946–17953. [CrossRef]
[PubMed]
29. Uddin, M.N.; Horvat, D.; Childs, E.W.; Puschett, J.B. Marinobufagenin causes endothelial cell monolayer
hyperpermeability by altering apoptotic signaling. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2009, 296,
R1726–R1734. [CrossRef] [PubMed]
30. Ing, N.H.; Berghman, L.; Abi-Ghanem, D.; Abbas, K.; Kaushik, A.; Riggs, P.K.; Puschett, J.B. Marinobufagenin
regulates permeability and gene expression of brain endothelial cells. Am. J. Physiol. Regul. Integr.
Comp. Physiol. 2014, 306, R918–R924. [CrossRef] [PubMed]
31. Afroze, S.H.; Sloan, J.; Grace-Ann, C.; Osuji, G.C.; Drever, N.; Pilkinton, K.; Zawieja, D.C.; Kuehl, T.;
Uddin, M.N. Cinobufotalin impedes Sw.71 cytotrophoblast cell line function via cell cycle arrest and
apoptotic signaling. Mol. Cell. Biochem. 2016, 422, 189–196. [CrossRef] [PubMed]
32. Kim, Y.J. Pathogenesis and promising non-invasive markers for preeclampsia. Obstet. Gynecol. Sci. 2013, 56,
2–7. [CrossRef] [PubMed]

Int. J. Environ. Res. Public Health 2017, 14, 517
14 of 15
33. Ehrig, J.C.; Horvat, D.; Allen, S.R.; Jones, R.O.; Kuehl, T.J.; Uddin, M.N. Cardiotonic steroids induce
anti-angiogenic and anti-proliferative profiles in first trimester extravillouscytotrophoblast cells. Placenta
2014, 35, 932–936. [CrossRef] [PubMed]
34. Gurusinghe, S.; Wallace, E.M.; Lim, R. The relationship between Activin A and anti-angiogenic factors in the
development of pre-eclampsia. Pregnancy Hypertens. 2014, 4, 3–6. [CrossRef] [PubMed]
35. Maynard, S.; Karumanchi, S. Angiogenic factors and preeclampsia. MBG is elevated in both animal models
of volume expansion and patients with preE. Semin. Nephrol. 2001, 31, 33–46. [CrossRef] [PubMed]
36. Levine, R.; Maynard, S.; Qian, C.; Lim, K.H.; England, L.J.; Yu, K.F.; Schisterman, E.F.; Thadhani, R.;
Sachs, B.P.; Epstein, F.H.; et al. Circulating angiogenic factors and the risk of preeclampsia. N. Engl. J. Med.
2004, 350, 672–683. [CrossRef] [PubMed]
37. Agarwal, I.; Karumanchi, S.A. Preeclampsia and the anti-angiogenic state. Pregnancy Hypertens. 2011, 1,
17–21. [CrossRef] [PubMed]
38. Maynard, S.E.; Min, J.Y.; Merchan, J.; Lim, K.H.; Li, J.; Mondal, S.; Libermann, T.A.; Morgan, J.P.; Sellke, F.W.;
Stillman, I.E.; et al. Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial
dysfunction, hypertension, and proteinuria in preeclampsia. J. Clin. Investig. 2003, 111, 649–658. [CrossRef]
[PubMed]
39. Cerdeira, A.S.; Karumanchi, S.A. Angiogenic factors in preeclampsia and related disorders. Cold Spring Harb.
Perspect. Med. 2012, 2, 1–17. [CrossRef] [PubMed]
40. Verlohren, S.; Herraiz, I.; Lapaire, O.; Schlembach, D.; Moertl, M.; Zeisler, H.; Calda, P.; Holzgreve, W.;
Galindo, A.; Engels, T.; et al. The sFlt-1/PlGF ratio in different types of hypertensive pregnancy disorders
and its prognostic potential in preeclamptic patients. Am. J. Obstet. Gynecol. 2012, 206, 58.e1–58.e8. [CrossRef]
[PubMed]
41. Stepan, H.; Herraiz, I.; Schlembach, D.; Verlohren, S.; Brennecke, S.; Chantraine, F.; Klein, E.; Lapaire, O.;
Llurba, E.; Ramoni, A.; et al. Implementation of the sFlt-1/PlGF ratio for prediction and diagnosis of
pre-eclampsia in singleton pregnancy: Implications for clinical practice. Ultrasound Obstet. Gynecol. 2015, 45,
241–246. [CrossRef] [PubMed]
42. Nikitina, E.R.; Mikhailov, A.V.; Nikandrova, E.S.; Frolova, E.V.; Fadeev, A.V.; Shman, V.V.; Shilova, V.Y.;
Tapilskaya, N.I.; Shapiro, J.I.; Fedorova, O.V.; et al. In preeclampsia endogenous cardiotonic steroids induce
vascular fibrosis and impair relaxation of umbilical arteries. J. Hypertens. 2011, 29, 769–776. [CrossRef]
[PubMed]
43. Khansari, M.E.; Johnson, C.R.; Basaran, I.; Nafis, A.; Wang, J.; Leszczynski, J.; Hossain, M.A. Synthesis
and anion binding studies of tris(3-aminopropyl)amine-based tripodal urea and thiourea receptors: Proton
transfer-induced selectivity for hydrogen sulfate over sulfate. RSC Adv. 2015, 5, 17606–17614. [CrossRef]
[PubMed]
44. Hossain, M.A. Inclusion complexes of halide anions with macrocyclic receptors. Curr. Org. Chem. 2008, 12,
1231–1256. [CrossRef]
45. Busschaert, N.; Wenzel, M.; Light, M.E.; Iglesias-Hernandez, P.; Perez-Tomas, R.; Gale, P.A. Structure-activity
relationships in tripodal transmembrane anion transporters: The effect of fluorination. J. Am. Chem. Soc.
2011, 133, 14136–14148. [CrossRef] [PubMed]
46. Haque, S.A.; Saeed, M.A.; Jahan, A.; Wang, J.; Leszczynski, J.; Hossain, M.A. Experimental and theoretical
aspects of anion complexes with a thiophene-based cryptand. Comments Inorg. Chem. 2016, 36, 305–326.
[CrossRef] [PubMed]
47. Rhaman, M.M.; Alamgir, A.; Wong, B.M.; Powell, D.R.; Hossain, M.A. A highly efficient dinuclear Cu(II)
chemosensor for colorimetric and fluorescent detection of cyanide in water. RSC Adv. 2014, 4, 54263–54267.
[CrossRef] [PubMed]
48. Rhaman, M.M.; Fronczek, F.R.; Powell, D.; Hossain, M.A. Colourimetric and fluorescent detection of oxalate
in water by a new macrocycle-based dinuclear nickel complex: A remarkable red shift of fluorescence band.
Dalton Trans. 2014, 43, 4618–4621. [CrossRef] [PubMed]
49. Rhaman, M.M.; Ahmed, L.; Wang, J.; Powell, D.; Leszczynski, J.; Hossain, M.A. Encapsulation and selectivity
of sulfate with a furan-based hexaaza macrocyclic receptor in water. Org. Biomol. Chem. 2014, 12, 2045–2048.
[CrossRef] [PubMed]
50. Khansari, M.E.; Wallace, K.D.; Hossain, M.A. Synthesis and anion recognition studies of a
dipodalthiourea-based sensor for anions. Tetrahedron Lett. 2014, 55, 438–440. [CrossRef]

Int. J. Environ. Res. Public Health 2017, 14, 517
15 of 15
51. Pramanik, A.; Powell, D.R.; Wong, B.M.; Hossain, M.A. Spectroscopic, structural, and theoretical studies of
halide complexes with a urea-based tripodal receptor. Inorg. Chem. 2012, 51, 4274–4284. [CrossRef] [PubMed]
52. Horvat, D.; Khansari, M.E.; Pramanik, A.; Beeram, M.R.; Kuehl, T.J.; Hossain, M.A.; Uddin, M.N. A synthetic
thiourea-based tripodal receptor that impairs the function of human first trimester cytotrophoblast cells.
Int. J. Environ. Res. Public Health 2014, 11, 7456–7469. [CrossRef] [PubMed]
53. Penniyaynen, V.A.; Kipenko, A.V.; Lopatina, E.V.; Bagrov, A.Y.; Krylov, B.V. The effect of marinobufagenin on
the growth and proliferation of cells in the organotypic culture. Dokl. Biol. Sci. 2015, 462, 164–166. [CrossRef]
[PubMed]
54. Afroze, S.H.; Munshi, M.K.; Martínez, A.K.; Uddin, M.N.; Gergely, M.; Szynkarski, C.; Guerrier, M.;
Nizamutdinov, D.; Dostal, D.; Glaser, S. Activation of the renin-angiotensin system stimulates biliary
hyperplasia during cholestasis induced by extrahepatic bile duct ligation. Am. J. Physiol. Gastrointest.
Liver Physiol. 2015. [CrossRef] [PubMed]
55. Sadoshima, J. Cytokine actions of angiotensin II. Circ. Res. 2000, 86, 1187–1189. [CrossRef] [PubMed]
56. Ehrig, J.C.; Afroze, S.H.; Reyes, M.; Allen, S.R.; Drever, N.S.; Pilkinton, K.A.; Kuehl, T.J.; Uddin, M.N. A p38
mitogen-activated protein kinase inhibitor attenuates cardiotonic steroids-induced apoptotic and stress
signaling in a Sw-71 cytotrophoblast cell line. Placenta 2015, 36, 1276–1282. [CrossRef] [PubMed]
57. Berg, C.J.; Atrash, H.K.; Koonin, L.M.; Tucker, M. Pregnancy-related mortality in the United States, 1987–1990.
Obstet. Gynecol. 1996, 88, 161–167. [CrossRef]
58. Pridjian, G.; Puschett, J.B. Preeclampsia. Part 1: Clinical and pathophysiologic considerations.
Obstet. Gynecol. Surv. 2002, 57, 598–618. [CrossRef] [PubMed]
©
2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).